The Use of Flow Cytometry to Study Neutrophils After Ischemic Stroke and Reperfusion

2.50
Hdl Handle:
http://hdl.handle.net/10755/157564
Type:
Presentation
Title:
The Use of Flow Cytometry to Study Neutrophils After Ischemic Stroke and Reperfusion
Abstract:
The Use of Flow Cytometry to Study Neutrophils After Ischemic Stroke and Reperfusion
Conference Sponsor:Western Institute of Nursing
Conference Year:2009
Author:Morrison, Helena, BSN
P.I. Institution Name:The University of Arizona, College of Nursing
Title:Doctoral Student
Contact Address:1305 N. Martin, PO Box 210203, Tucson, AZ, 85721, USA
Contact Telephone:520-626-3218
Co-Authors:Leslie S. Ritter, PhD, RN, Associate Professor
Purpose: The purpose of this paper is to discuss the use of flow cytometry to examine neutrophil activation within the first minutes of reperfusion after ischemic stroke. Background: Activated neutrophils contribute significantly to inflammation during disease and injury. A characteristic of neutrophil activation is their surface expression of the CD11b/CD18 adhesion molecule. Neutrophil CD11b binds to its ligand intracellular adhesion molecule one (ICAM-1) on activated endothelium, facilitating firm adhesion to the vasculature. Neutrophil adhesion to the vasculature during reperfusion is associated with increased tissue injury via multiple mechanisms: the no reflow phenomena caused by cell trapping in the microvasculature, the release of reactive oxygen species (ROS) and proteases from neutrophils, and the systemic and local release of pro-inflammatory cytokines. Using microcirculatory techniques, we have previously shown that neutrophils adhere to the microcirculation during the first minutes of reperfusion after ischemic stroke. However, neutrophil activation, measured by surface expression of CD11b and intracellular ROS production, has not been investigated during ischemic stroke and early reperfusion. Methods: Flow cytometry was used to quantify neutrophil activation during early reperfusion after ischemic stroke. Two groups of male Sprague-Dawley rats (275-325g) were used: one group underwent middle cerebral artery occlusion (4 hours) and reperfusion (MCAOR) via the filament method and the other group underwent sham surgery. One milliliter of blood was drawn into 0.1 ml citrate before the MCAOR or sham surgery and again at fifteen minutes of reperfusion (or at the similar time point for the sham animals). Whole blood was incubated with PE-Cy5 conjugated anti-rat mAb to CD45 for neutrophil identification. To measure neutrophil expression of CD11b, the same blood was incubated with FITC-conjugated anti-rat mAb to CD11b. To measure ROS production, CD45 labeled blood was incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCF). Additional aliquots of blood were also incubated with neutrophil activator fMLP as a positive control. Flow cytometry data are reported as total fluorescence intensity (TFI). A repeated measure ANOVA was used to compare CD11b expression and ROS production between groups (MCAOR, Sham, and MCAOR/fMLP) and over time (pre-ischemia and reperfusion). Results: When compared to the pre-ischemia time and Sham group, the MCAOR groups at fifteen minutes of reperfusion demonstrated a significant increase in neutrophil surface expression of CD11b (p<0.007, n=5), a significant increase in fMLP stimulated CD11b expression (p=0.002, n=5), a trend toward an increase in neutrophil ROS production (p=0.34, n=5), and a non-significant increase in fMLP stimulated ROS production after reperfusion (p=0.11, n=5). Conclusions: These data indicate that in a rodent model, neutrophils are activated as early as 15 minutes of reperfusion following cerebral ischemia, evidenced by a significant increase in surface expression of CD11b adhesion molecule. Continued investigation of complex blood cell inflammatory responses during ischemic stroke and reperfusion could be elucidated with the use of this method. As seen in our study, flow cytometry is a very useful and exciting method for use in nursing research, analyzing a variety of cellular alterations.
Repository Posting Date:
26-Oct-2011
Date of Publication:
17-Oct-2011
Sponsors:
Western Institute of Nursing

Full metadata record

DC FieldValue Language
dc.typePresentationen_GB
dc.titleThe Use of Flow Cytometry to Study Neutrophils After Ischemic Stroke and Reperfusionen_GB
dc.identifier.urihttp://hdl.handle.net/10755/157564-
dc.description.abstract<table><tr><td colspan="2" class="item-title">The Use of Flow Cytometry to Study Neutrophils After Ischemic Stroke and Reperfusion</td></tr><tr class="item-sponsor"><td class="label">Conference Sponsor:</td><td class="value">Western Institute of Nursing</td></tr><tr class="item-year"><td class="label">Conference Year:</td><td class="value">2009</td></tr><tr class="item-author"><td class="label">Author:</td><td class="value">Morrison, Helena, BSN</td></tr><tr class="item-institute"><td class="label">P.I. Institution Name:</td><td class="value">The University of Arizona, College of Nursing</td></tr><tr class="item-author-title"><td class="label">Title:</td><td class="value">Doctoral Student</td></tr><tr class="item-address"><td class="label">Contact Address:</td><td class="value">1305 N. Martin, PO Box 210203, Tucson, AZ, 85721, USA</td></tr><tr class="item-phone"><td class="label">Contact Telephone:</td><td class="value">520-626-3218</td></tr><tr class="item-email"><td class="label">Email:</td><td class="value">hmorrison@nursing.arizona.edu</td></tr><tr class="item-co-authors"><td class="label">Co-Authors:</td><td class="value">Leslie S. Ritter, PhD, RN, Associate Professor</td></tr><tr><td colspan="2" class="item-abstract">Purpose: The purpose of this paper is to discuss the use of flow cytometry to examine neutrophil activation within the first minutes of reperfusion after ischemic stroke. Background: Activated neutrophils contribute significantly to inflammation during disease and injury. A characteristic of neutrophil activation is their surface expression of the CD11b/CD18 adhesion molecule. Neutrophil CD11b binds to its ligand intracellular adhesion molecule one (ICAM-1) on activated endothelium, facilitating firm adhesion to the vasculature. Neutrophil adhesion to the vasculature during reperfusion is associated with increased tissue injury via multiple mechanisms: the no reflow phenomena caused by cell trapping in the microvasculature, the release of reactive oxygen species (ROS) and proteases from neutrophils, and the systemic and local release of pro-inflammatory cytokines. Using microcirculatory techniques, we have previously shown that neutrophils adhere to the microcirculation during the first minutes of reperfusion after ischemic stroke. However, neutrophil activation, measured by surface expression of CD11b and intracellular ROS production, has not been investigated during ischemic stroke and early reperfusion. Methods: Flow cytometry was used to quantify neutrophil activation during early reperfusion after ischemic stroke. Two groups of male Sprague-Dawley rats (275-325g) were used: one group underwent middle cerebral artery occlusion (4 hours) and reperfusion (MCAOR) via the filament method and the other group underwent sham surgery. One milliliter of blood was drawn into 0.1 ml citrate before the MCAOR or sham surgery and again at fifteen minutes of reperfusion (or at the similar time point for the sham animals). Whole blood was incubated with PE-Cy5 conjugated anti-rat mAb to CD45 for neutrophil identification. To measure neutrophil expression of CD11b, the same blood was incubated with FITC-conjugated anti-rat mAb to CD11b. To measure ROS production, CD45 labeled blood was incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCF). Additional aliquots of blood were also incubated with neutrophil activator fMLP as a positive control. Flow cytometry data are reported as total fluorescence intensity (TFI). A repeated measure ANOVA was used to compare CD11b expression and ROS production between groups (MCAOR, Sham, and MCAOR/fMLP) and over time (pre-ischemia and reperfusion). Results: When compared to the pre-ischemia time and Sham group, the MCAOR groups at fifteen minutes of reperfusion demonstrated a significant increase in neutrophil surface expression of CD11b (p&lt;0.007, n=5), a significant increase in fMLP stimulated CD11b expression (p=0.002, n=5), a trend toward an increase in neutrophil ROS production (p=0.34, n=5), and a non-significant increase in fMLP stimulated ROS production after reperfusion (p=0.11, n=5). Conclusions: These data indicate that in a rodent model, neutrophils are activated as early as 15 minutes of reperfusion following cerebral ischemia, evidenced by a significant increase in surface expression of CD11b adhesion molecule. Continued investigation of complex blood cell inflammatory responses during ischemic stroke and reperfusion could be elucidated with the use of this method. As seen in our study, flow cytometry is a very useful and exciting method for use in nursing research, analyzing a variety of cellular alterations.</td></tr></table>en_GB
dc.date.available2011-10-26T19:59:23Z-
dc.date.issued2011-10-17en_GB
dc.date.accessioned2011-10-26T19:59:23Z-
dc.description.sponsorshipWestern Institute of Nursingen_GB
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